Application of norharman in preparation of quorum sensing inhibitor and bacterial strain
A demethylation and quorum sensing technology, applied in the direction of bacteria, local antibacterial agents, and microbial-based methods, to achieve the effects of reduced pyocyanin production, enhanced inhibitory effect, and inhibited biofilm formation
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Embodiment 1
[0042] Embodiment 1, strain 40DY182 isolating, extracting and identifying
[0043] 1. Preliminary screening of strain 40DY182
[0044] The soil samples at 5591m below the western Pacific Ocean were collected, and the microbial strains were isolated by the spread plate method. Take 0.5g soil sample, add 4.5mL sterile water, record it as sample 1, mix it and let it stand for 2h. Take 0.5mL sample 1 and add 4.5mL sterile water, record it as sample 2, mix it and let stand for 2h. Take 0.5mL sample 2 and add 4.5mL sterile water, record it as sample 3, mix it and let stand for 2h. Samples 1, 2, and 3 were respectively coated and inoculated in YP seawater solid medium, MM and LB solid medium, cultured in a constant temperature incubator at 30°C for 7 days, and 3 plates were saved for each strain for later use. Observe the strain morphology and record it.
[0045] 2. Re-screening
[0046] Pick the formed and single bacterial colony in the primary screening to the YP seawater soli...
Embodiment 2
[0052] Embodiment 2, acquisition and identification of strain C218
[0053] Strain C218 was provided by Li Jiangtao, the Second Affiliated Hospital of Zhejiang University School of Medicine.
[0054] According to the 16S rRNA sequence (as shown in SEQ ID NO.2), combined with ecological characteristics, the strain C218 was identified as Pseudomonas aeruginosa (P.aeruginosa), named Pseudomonas aeruginosa (Pseudomonas aeruginosa) C218, and preserved in Guangdong Microbial Culture Collection Center, date of deposit is May 13, 2020, collection number GDMCC No: 61027, address: 5th Floor, Building 59, Compound, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Institute of Microbiology, postal code 510075.
[0055] The measured gene sequence (SEQ ID NO.2) is:
[0056] CCTTGCGGTTAGACTAGCTACTTCTGGAGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGACATTCTGATTCACGATTACTAGCGATTCCGACTTCACGCAGTCGAGTTGCAGACTGCGATCCGGACTACGATCGGTTTTATGGGATTAGCTCCACCTCGCGGCTTGGCAACCCTTTGTACCGACCATT...
Embodiment 3
[0057] Embodiment 3, the extraction identification of compound norharmane
[0058] 1. Preparation of crude extract
[0059] Inoculate marine bacteria 40DY182 into YP seawater solid slant medium, culture overnight in a 30°C constant temperature incubator to obtain slant bacteria; YP seawater solid medium: yeast extract 1g / L, peptone 5g / L, sea salt 22.5g / L L, agar 15g / L, solvent is deionized water, sterilized at 121°C for 20min;
[0060] Then inoculate the slant bacteria into YP seawater liquid medium, and culture in a constant temperature shaker at 30°C with 180rpm shaking overnight to obtain seed liquid; YP seawater solid medium: yeast extract 1g / L, peptone 5g / L, sea salt 22.5 g / L, agar 15g / L, solvent is deionized water, sterilized at 121°C for 20min;
[0061] The seed solution was inoculated into a 100L fermenter equipped with YP seawater liquid medium at an inoculum volume concentration of 2.5%, and fermented and cultivated at 30°C and a pressure of 0.1Mpa for 2 days. One ...
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